WO2022087856A1

WO2022087856A1 - Antigen presenting system of novel coronavirus vaccine using attenuated salmonella for secreting and expressing rbd domain protein, and use thereof

Info

Publication number: WO2022087856A1
Application number: PCT/CN2020/124173
Authority: WO
Inventors: 华子春; 吴乐阳; 李霖
Original assignee: 南京吉芮康生物科技研究院有限公司; 江苏靶标生物医药研究所有限公司
Priority date: 2020-10-26
Filing date: 2020-10-27
Publication date: 2022-05-05
Also published as: CN114480463A

Abstract

An antigen presenting system of a novel coronavirus vaccine using attenuated salmonella for secreting and expressing an RBD domain protein, and the use thereof. The antigen presentation system is used for preventing novel coronavirus (SARS-CoV-2). By means of constructing a controllable and stable expression plasmid that secretes and expresses the RBD domain protein and engineering attenuated salmonella, an antigenic protein can be efficiently delivered to an antigen-presenting cell via an oral route and by means of the specific secretion system thereof after administration. The delivered antigenic protein can be effectively processed and presented by antigen-presenting cells, thereby ultimately realizing the activation/regulation of the immune system, producing antibodies, and exhibiting the function of a vaccine.

Description

A novel coronavirus vaccine antigen presentation system secreting and expressing RBD domain protein by attenuated Salmonella and its application

technical field

The invention relates to the field of biotechnology, in particular to a novel coronavirus vaccine antigen presentation system for secreting and expressing RBD domain proteins by attenuated Salmonella and its application.

Background technique

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the most serious challenge humanity has faced in a century, as of September 1, 2020, More than 27 million people have been infected and more than 800,000 people have died. The novel coronavirus shares about 82% genome similarity with the SARS coronavirus (SARS-CoV) that broke out in 2003, and both coronaviruses also share the same cellular receptor, angiotensin-converting enzyme 2 (ACE2) ( Lan J et al, 2020, Nature 581:215-220.). Despite these similarities, 2019-nCoV spreads more widely, more rapidly, and more lethally than SARS virus (Zhu N et al., 2020, N Engl J Med, 382:727-733.). Therefore, in order to effectively prevent the spread of the new coronavirus, it is urgent to develop a safe, efficient and inexpensive SARS-CoV-2 vaccine. During the early development of a vaccine against SARS-CoV, researchers found that antibodies against the viral spike protein (S protein) were highly effective in neutralizing the virus and preventing infection (Yang ZY et al., 2005, Proc Natl Acad Sci U S A 102:797-801.), therefore targeting the S protein of SARS-CoV-2, especially the RBD region in this protein, is the primary target for current antiviral drug and vaccine development (Walls AC et al., 2020, Cell 181 :281-292.e6.).

At this stage, a number of research institutes and pharmaceutical companies are rapidly developing new coronavirus vaccines, involving live attenuated vaccines, recombinant viral vector vaccines, inactivated virus vaccines, protein subunit vaccines, virus-like particle (VLP) vaccines and nucleic acid vaccines etc. (Jeyanathan M et al., 2020, Nat Rev Immunol 20:615-632.). Although live attenuated vaccines show high protective power, they are also accompanied by high risks; inactivated vaccines, recombinant virus vaccines and nucleic acid vaccines face problems such as high cost and inconvenience in vaccination; direct vaccination of traditional recombinant protein vaccines cannot be very effective. Good stimulation of cellular immune response, but can not provide better protection, often requires the addition of additional adjuvants (Guy B et al., 2007, Nat Rev Microbiol 5:505-517.). So far, all vaccines developed at home and abroad are administered by injection. At present, many countries have reported the side effects of the new crown vaccine, and even reports of deaths. Injection is the most direct route of administration, but it is also the most risky and costly of all routes of administration. So far, there is a lack of other more convenient and safe administration methods or routes of administration in the development of new crown vaccines.

The working principle of the vaccine is: when the vaccine antigen is inoculated into the animal body, the immune system of the animal body is stimulated, and the antigen-presenting cells of the animal body process, process and present the vaccine to specific lymphocytes (T and B lymphocytes), Then lymphocytes recognize, activate, proliferate, and differentiate to the vaccine and finally produce immune effector molecules (antibodies and cytokines) and immune effector cells, and finally remove the antigen from the animal body. This process is called immune response. It can be seen that the current international method of injecting antigens in the development of new coronavirus vaccines is likely to cause some side effects of the injected antigens in systemic tissues. The process of exerting the vaccine to induce immunity is actually through antigenic extraction produced by cells. Therefore, if the production of antigens is restricted to antigen-presenting cells, it is possible to limit the possible side effects of antigens in systemic tissues and achieve a safer goal.

Oral vaccines based on attenuated strains are widely used in the development of vaccines for various infectious diseases due to their advantages of simple vaccination and low price. Attenuated Salmonella is a widely used bacterial oral vaccine carrier, as well as a natural mucosal immune adjuvant, antigen expression and delivery tool. Genetically engineered recombinant vaccine strains enter the body through M cells in the gut after oral administration (Jensen VB et al., 1998, Infect Immun 66:3758-3766.), and the strains entering the in vivo environment can be processed by antigen-presenting cells (APCs). ) is rapidly phagocytosed, at this time, if the secretory system of the bacteria is used, the antigenic protein can be effectively secreted into the interior of APC cells, and further effectively decomposed into polypeptide fragments and passed through the histocompatibility complex MHC-I or MHC-II. The pathway is displayed to T-help cells to stimulate the body to generate cellular, humoral and mucosal immune responses against antigenic molecules (Mei Y et al., 2017, Cancer Immunol Res 5:503-514.).

However, the development of an efficient and highly applicable oral attenuated Salmonella vaccine for the prevention of 2019-nCoV still faces the following technical difficulties: a) maintaining the phenotypic stability of recombinant bacteria and using attenuated bacteria as an expression tool for foreign antigens , it is necessary to establish a suitable expression strategy to optimize the antigen expression amount and plasmid compatibility, otherwise there will be problems such as excessive strain toxicity, serious loss of engineering plasmids and loss of immune effect; b) antigenic proteins are secreted into cells by bacteria Because most bacteria are encapsulated by a membrane-coated vesicle structure (SCV) after entering antigen-presenting cells, this structure limits the effective presentation of antigen molecules to a great extent (Zhang XL et al., 2008, Cell Mol Immunol 5: 91-7.), if the real effective presentation cannot be achieved, the preventive effect of the vaccine will be greatly reduced; c) What kind of promoter is selected so that the antigen molecule is only in the antigen presenting cell Expression, but not in blood and normal tissues, provides the safety of antigen molecules. d) The optimality of virus epitope screening and region selection. Due to the large molecular weight and complex structure of the new coronavirus S protein (Hsieh CL et al., 2020, Science 369: 1501-1505.), it is necessary to target different domains of the virus. Epitopes are screened to avoid overly complex protein structures that cannot be efficiently secreted, and to induce a better immune response by presenting as many effective epitopes as possible.

Therefore, in order to obtain an efficient SARS-CoV-2 vaccine based on the attenuated Salmonella secretion expression system (SARS-CoV-2), a safe, efficient and stable Salmonella secretion expression vector must be constructed to achieve the expression of antigen molecules in Salmonella and It can be effectively secreted into antigen-presenting cells to efficiently induce immune response of the body, which is the problem to be solved by the present invention.

Because different proteins have different primary sequences, the hydrophobic properties and charge distributions of different proteins are very different, and the different primary sequences of different proteins cause different proteins to have different spatial structures or higher-order structures, resulting in the spatial structure of proteins. The types and physical and chemical properties of protein surfaces vary widely. Therefore, it is extremely difficult to achieve efficient, stable and secreted expression of different antigenic proteins in Salmonella, which cannot be predicted or inferred. It needs to be studied and explored one by one according to different proteins, and creative work is required.

SUMMARY OF THE INVENTION

The main purpose of the present invention is to construct an efficient attenuated Salmonella expression and secretion vector, and to screen out the combination of novel coronavirus antigenic epitopes that can induce the best immune response, so as to establish a stable expression and high protective efficiency for the attenuated Salmonella. Laying the groundwork for a novel coronavirus oral vaccine for delivery vehicles.

In order to achieve the above purpose, the present invention provides the following technical solutions: a novel coronavirus vaccine antigen presentation system of the present invention that attenuated Salmonella secretes and expresses an RBD domain protein, and the attenuated Salmonella secretes and expresses an RBD domain protein The novel coronavirus vaccine antigen presentation system includes the type III secretion system promoter and signal peptide sequence; it can realize the secretory expression of the novel coronavirus antigen in antigen-presenting cells through attenuated Salmonella, and induce the body to produce high titers in a mouse model Antibody.

Further, the intracellular inducible promoter of the antigen-presenting cell is used to regulate the secretion and expression of the antigen by the bacterial secretion signal, the Salmonella secretion and expression system is used to enable the antigen to be secreted, and the plasmid anti-loss element is added to improve the plasmid stability of the expression vector in Salmonella, Thereby, a highly efficient, stable, and intracellularly regulated antigen presenting system of attenuated Salmonella secreted and expressed by antigen presenting cells is obtained.

Further, the novel coronavirus vaccine antigen is a combination of novel coronavirus antigenic epitopes that have been screened and can induce the best immune response. Specifically, the combination of novel coronavirus antigenic epitopes that can induce the best immune response is SARS-CoV. -2 Spike protein (S protein) RBD domain, the gene sequence of the RBD domain is the nucleotide sequence shown in SEQ ID No. 6, which is located at amino acids 319-541 of the overall amino acid sequence of S protein.

Further, the intracellular inducible promoter of the antigen-presenting cell is the Salmonella sifB promoter; the gene sequence of the Salmonella sifB promoter is the nucleotide sequence shown in SEQ ID No.4.

Further, the intracellular inducible promoter of the antigen presenting cell is the Escherichia coli NirB promoter, or the Salmonella SseA promoter, or the Salmonella SseJ promoter; the gene sequence of the Escherichia coli sifB promoter is shown in SEQ ID No.1. The nucleotide sequence shown, the gene sequence of the Salmonella SseA promoter is the nucleotide sequence shown in SEQ ID No.2, and the gene sequence of the Salmonella SseJ promoter is the nucleotide sequence shown in SEQ ID No.3 acid sequence.

Further, the bacterial secretion signal secretion expression system is a Salmonella type III secretion expression system, and the type III secretion signal is the Salmonella virulence island 2 (SPI-2) effector protein SseJ signal peptide, and the Salmonella virulence island 2 (SPI-2) effector protein SseJ signal peptide. -2) The gene sequence of the effector protein SseJ signal peptide is the nucleotide sequence shown in SEQ ID No.5.

Further, the plasmid loss prevention element is an AT element sequence, and the gene sequence of the AT element sequence is the nucleotide sequence shown in SEQ ID No.8.

Further, the expression of vaccine antigen is achieved by attenuated Salmonella, said attenuated Salmonella is VNP20009, htrA gene-deficient attenuated Salmonella VNP20009 (Ah-1).

The application of the novel coronavirus vaccine antigen presentation system of the attenuated Salmonella secreting and expressing the RBD domain protein of the present invention, through oral administration of the attenuated virus vaccine antigen presentation system of the novel coronavirus containing the attenuated Salmonella secreting and expressing the RBD domain protein Salmonella induces the production of high titer antibodies in a mouse model.

The application of the novel coronavirus vaccine antigen presentation system of the attenuated Salmonella secreting and expressing the RBD domain protein of the present invention as a medicine for preparing novel coronavirus vaccines and preventing novel coronaviruses.

Beneficial effects: a novel, efficient, safe, intracellular expression of antigen-presenting cells, low-cost, convenient, novel coronavirus vaccine secretion expression vector that can be used for humans and animals and its application. By constructing a controllable, stable expression plasmid that secretes and expressing the RBD domain protein and engineering attenuated Salmonella, the present invention can present the antigen to the antigen through the oral route and the unique secretion system after administration. Cells efficiently deliver antigenic proteins. The delivered antigenic proteins can be efficiently processed and presented by antigen-presenting cells, and finally activate/regulate the immune system, produce antibodies, and play the role of vaccines.

Compared with the existing and new coronavirus vaccines under development, the features and innovations of the present invention are:

(1) The present invention provides a novel coronavirus vaccine antigen expression vector using Salmonella type III secretion system signal to guide the novel coronavirus vaccine antigen expression vector, realizes the secretory expression of the novel coronavirus antigen, and induces the body to produce high-titer antibodies in a mouse model.

(2) The present invention provides a novel oral bacteria-made novel coronavirus vaccine, which uses a novel attenuated Salmonella as a carrier, and the novel attenuated Salmonella itself can be used as an efficient adjuvant due to its weak pathogenicity. agent, eliminating the need for additional adjuvant addition.

(3) The present invention provides a low-cost oral bacteria-made novel coronavirus vaccine. The vaccine uses a novel attenuated Salmonella as a carrier. Due to the self-propagating property of the attenuated strain, the production cost is low and the preparation of the vaccine is greatly reduced. Time, simplifies the operation process, and makes the final product have a stronger price advantage.

(4) The present invention provides a novel coronavirus vaccine expressed intracellularly in antigen-presenting cells. The vaccine is only expressed intracellularly in antigen-presenting cells, which can not only effectively produce the immune effect of the vaccine, but also effectively prevent the antigen from The presence of normal tissue and possible side effects, thus enabling greater safety.

(5) The present invention provides a safe and controllable oral vaccine system, which uses a novel attenuated Salmonella as a carrier, and can achieve effective and rapid regression by taking conventional Salmonella-sensitive antibiotics.

Description of drawings

Figure 1 is a schematic diagram of the novel coronavirus RBD domain of the present invention and its binding to receptors;

1A is a schematic structural diagram of the position of the RBD protein domain of the novel coronavirus of the present invention in the S protein;

Figure 1B is a schematic diagram of the binding of the novel coronavirus RBD protein domain to its receptor ACE2 protein of the present invention, 1. ACE2 protein; 2. RBD protein domain; 3. Enlarged schematic diagram of the binding region between ACE2 and RBD protein domain.

2 is a schematic diagram of the construction of different new coronavirus RBD protein domain expression systems of the present invention;

1.Ah-JP-RBD plasmid, using J23100 promoter, pelB signal peptide, expressing RBD protein; 2.Ah-NS-RBD plasmid, using Salmonella NirB promoter, the gene sequence of said Salmonella NirB promoter is SEQ ID No Nucleotide sequence shown in .1; SseJ signal peptide, expresses RBD protein; 3.Ah-SS-RBD plasmid, uses SseA promoter, the gene sequence of described SseA promoter is the nucleus shown in SEQ ID No.2 nucleotide sequence; SseJ signal peptide, expressing RBD protein; 4.Ah-JJ-RBD plasmid, using SseJ promoter, the gene sequence of the SseJ promoter is the nucleotide sequence shown in SEQ ID No.3; SseJ signal Peptide, expresses RBD protein; 5.Ah-BJ-RBD plasmid, uses SifB promoter, the gene sequence of described SifB promoter is the nucleotide sequence shown in SEQ ID No.4; SseJ signal peptide, uses SifB promoter , expressing RBD protein.

3 is a graph showing the expression and secretion of the new coronavirus RBD protein of the recombinant attenuated Salmonella of different expression systems detected by WB of the present invention;

3A is a graph showing the expression and secretion of the new coronavirus RBD protein of the Ah-JP-RBD recombinant attenuated Salmonella of the present invention. 1. Collect the protein expression detection in the bacteria, 2. Collect the culture supernatant, and detect the secretion after protein expression. Ah-JP-RBD recombinant attenuated Salmonella can effectively express and secrete SARS-CoV-2 RBD protein;

Figure 3B is a graph showing the expression and secretion of the new coronavirus RBD proteins of four recombinant attenuated Salmonella species of the present invention, Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD, and Ah-BJ-RBD. a. Bacteria were cultured in LB liquid medium; b. Bacteria were co-cultured with macrophage RAW264.7. 1.Ah-NS-RBD recombinant attenuated Salmonella; 2.Ah-SS-RBD recombinant attenuated Salmonella; 3.Ah-JJ-RBD recombinant attenuated Salmonella; 4.Ah-BJ-RBD recombinant attenuated Salmonella. Ah-NS-RBD and Ah-SS-RBD recombinant attenuated Salmonella could not induce the expression of 2019-nCoV RBD protein in macrophage RAW264.7 cells, while Ah-JJ-RBD and Ah-BJ-RBD reduced Salmonella virulence can effectively induce the expression and secretion of SARS-CoV-2 RBD protein in macrophage RAW264.7 cells.

4 is a graph showing the intracellular expression and secretion of the novel coronavirus RBD protein of the recombinant attenuated Salmonella engineered bacteria detected by immunofluorescence of the present invention;

1. Recombinant attenuated Salmonella was loaded with plasmids without RBD-HA sequence but with the same other elements; 2. Ah-JJ-RBD recombinant attenuated Salmonella; 3. Ah-BJ-RBD recombinant attenuated Salmonella. DAPI stained nuclei, Ah-1 was stained with Salmonella fluorescent antibody (arrow), and RBD-HA was stained with corresponding fluorescent antibody (arrow).

Fig. 5 is the experimental schematic diagram of the recombinant bacteria immunized mice of the present invention;

Figure 6 is an evaluation diagram of the antibody production of the new coronavirus RBD protein induced by three recombinant attenuated Salmonella species of the present invention, Ah-JP-RBD, Ah-JJ-RBD and Ah-BJ-RBD;

The blank control group was Ah-NC recombinant attenuated Salmonella; 2.Ah-JP-RBD recombinant attenuated Salmonella; 3.Ah-JJ-RBD recombinant attenuated Salmonella; 4.Ah-BJ-RBD recombinant attenuated Salmonella. The three recombinant attenuated Salmonella species can effectively induce the production of corresponding antibodies against the new coronavirus RBD protein in mice. Among them, the effect of Ah-BJ-RBD recombinant attenuated Salmonella in inducing antibodies is significantly better than that of the other three recombinant bacteria, Ah-BJ - Compared with Ah-NC, Ah-JP-RBD and Ah-JJ-RBD recombinant attenuated Salmonella, the ELISA RBD protein titers of RBD attenuated Salmonella increased by 6.03, 3.55 and 2.19 times respectively, indicating that this system is more efficient .

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Take the RBD protein region of the new coronavirus SARS-COV-2 S protein as the delivery antigen as an example. The attenuated Salmonella used in the examples is the htrA-deficient VNP20009 attenuated strain (referred to as Ah-1). If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer.

Example 1

Construction of Antigen Protein Novel Coronavirus Protein RBD Delivery Plasmid

RBD is a partial structural region of the new coronavirus S protein (Figure 1). Structural analysis shows that RBD protein plays a key role in the binding of S protein to ACE2 (angiotensinase 2). Considering the large structural region of S, after extensive and multi-angle bioinformatics analysis and mutual comparison, it is predicted that the RBD located at positions 319-541 of the overall amino acid sequence of S protein contains more epitope sites. After a lot of experimental research, we tried various reported commonly used bacterial secretion systems, secretion signal peptides and matching promoters. In the Ah-JP-RBD plasmid of constitutive expression and secretion system, after screening, the present invention selects and uses strong constitutive promoter J23100, pelB signal peptide. For inducible expression type III secretion expression plasmids Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD, Ah-BJ-RBD plasmids, the present invention attempts to use hypoxic promoter NirB, type III secretion The system-related promoters SseA, SseJ, SifB, and the four plasmids all use the type III secretion system-related signal peptide SseJ. The N ends of the above-mentioned 5 plasmids are coupled with the antigen molecule NTD protein sequence by means of Linker, and the gene sequence of the Linker sequence is the nucleotide sequence shown in SEQ ID No.7. To facilitate subsequent detection, the RBD was conjugated to the HA tag (Figure 2).

NirB, sseA, sseJ and sifB promoters, sseJ, PelB signal peptide, and RBD in the S protein region of SARS-COV-2 were all obtained by PCR.

The relevant primer sequence is: PNirB P1, and the gene sequence of the PNirB P1 primer is the nucleotide sequence shown in SEQ ID No.9.

PNirB P2; the gene sequence of the PNirB P2 primer is the nucleotide sequence shown in SEQ ID No.10.

PsseA P1; the gene sequence of the PsseA P1 primer is the nucleotide sequence shown in SEQ ID No.11.

PsseA P2; the gene sequence of the PsseA P2 primer is the nucleotide sequence shown in SEQ ID No.12.

PsifB P1:5'-caaaatcccttataagaattctgccctaccgctaaacatc-3'; the gene sequence of the PsifB P1 primer is the nucleotide sequence shown in SEQ ID No.13.

PsifB P2: 5'-tgtccaacactcaatggcatccacaagtgattatatgata-3'; the gene sequence of the PsifB P2 primer is the nucleotide sequence shown in SEQ ID No.14.

SseJ P1:5'-tatcatataatcacttgtggatgccattgagtgttggaca-3'; the gene sequence of the SseJ P1 primer is the nucleotide sequence shown in SEQ ID No.15.

SseJ P2: 5'-gccttcagtggaataatgatgagctataaaactttctaac-3'; the gene sequence of the SseJ P2 primer is the nucleotide sequence shown in SEQ ID No.16.

PsseJ-sseJ P1:5'-caaaatcccttataagaatttcacataaaacactagcact-3'; the gene sequence of the PsseJ-sseJ P1 primer is the nucleotide sequence shown in SEQ ID No.17.

PsseJ-sseJ P2: 5'-GCCttcagtggaataatgatgagctataaaactttctaac-3'; the gene sequence of the PsseJ-sseJ P2 primer is the nucleotide sequence shown in SEQ ID No.18.

50ng of Salmonella genomic DNA was used as template.

RBD P1:5'-agcggaggtggaggcagcccgaacatcaccaacctg-3'; the gene sequence of the RBD P1 primer is the nucleotide sequence shown in SEQ ID No.19.

RBD P2: 5'-tctggaacatcgtatgggtacggcgcgtgcagcagttc-3'; the gene sequence of the RBD P2 primer is the nucleotide sequence shown in SEQ ID No.20.

Using commercial plasmid MC_0101082 as template;

Vec P1:5'-tacccatacgatgttccagattacg-3'; the gene sequence of the Vec P1 primer is the nucleotide sequence shown in SEQ ID No.21.

Vec P2:5'-gctgcctccacctccgctgc-3'; the gene sequence of the Vec P2 primer is the nucleotide sequence shown in SEQ ID No.22.

The plasmid pQE30 with AT element in our laboratory was used as the template. The Linker sequence between the signal peptide and the target protein was obtained by thermal annealing self-linking method. After each fragment was obtained by PCR, the corresponding fragments were assembled by homologous recombination method, and finally various protein expression and secretion vectors of type III secretion system were obtained, including Ah-JP-RBD, Ah-NS-RBD, Ah-SS- RBD, Ah-JJ-RBD, Ah-BJ-RBD.

Example 2

Electroporation Transformation of Recombinant Attenuated Salmonella:

Preparation of Salmonella electrotransformation competence: inoculate fresh attenuated Salmonella into 200mL LB medium, culture on a shaker at 37°C until the OD value is between 0.4-0.6, centrifuge at 5000rpm, collect the bacteria for 5min, and wash once with sterile double-distilled water Then, centrifuge at 5000 rpm for 5 min, wash the cells with sterilized 10% glycerol 3-5 times, centrifuge at 5000 rpm for 5 min, resuspend with 500 μL of 10% glycerol, and dispense 50 μL/tube for electroporation. The recombinant vaccine DNA vector was transformed into attenuated Salmonella by electroporation: under sterile conditions, 0.5-5 μg of the constructed recombinant vector was added to the electroporation competent, and after mixing, it was transferred to a 2 mm electroporation cup. For electric shock, the electric transfer conditions are 1.8kV, 25μF, 500Ω. After electroporation, it was coated on a kanamycin plate for screening, and the grown colonies were the constructed recombinant bacteria, and the monoclonal strains were selected for sequencing verification.

Example 3

Detection of effective secretion of antigenic proteins

The obtained recombinant attenuated Salmonella was cultured in kanamycin-resistant liquid LB medium to an OD600 of 0.8-1.0, the cells were collected and the OD600 value was adjusted to about 1.0 with PBS. Place 4 degrees for spare. The macrophage cell line RAW264.7 was induced with 100 ng/mL LPS to obtain M1-type macrophages for 24 hours (referred to as RAW264.7(M1) below). The obtained RAW264.7 (M1) was co-cultured at 1:10 with four recombinant attenuated Salmonella species, Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD and Ah-BJ-RBD obtained above at 1:10 for 90 minutes, the supernatant was discarded, washed 2-3 times with PBS, and cultured in cell culture medium (10% serum, without double antibody) supplemented with 100 ng/mL gentamicin for 6 hours. Collect cells and collect total cell protein by thermal lysis method, that is, resuspend cells in 100 μL PBS, add 25 μL 5X Loading Buffer, and lyse at 100 degrees for 10-15 minutes. After centrifugation at 9,000 rpm for 5 minutes to collect the four recombinant bacteria in LB, the same method was used to collect the total protein in the bacteria.

For Ah-JP-RBD recombinant attenuated Salmonella, the inoculated cells were expanded and cultured in 50 mL of kana-resistant liquid LB to an OD600 of about 1.0. The total protein in the supernatant was collected by TCA (trichloroacetic acid)-acetone precipitation method. Briefly, the supernatant was transferred to a 50 ml centrifuge tube, and centrifuged at 15,000 g, 4 degrees, for 10 min using an ultracentrifuge. Take the supernatant and transfer it to a new 50ml centrifuge tube, add 10% TCA, vortex to mix well, and let stand on ice for 30min. Centrifuge again at 7,000g for 20min at 4°C, resuspend the pellet in 300μl PBS, and transfer it to a 1.5ml sterile EP tube. Add 1.2ml of pre-cooled acetone (placed in advance -20), 17,000g, 4 degrees, centrifugation for 20min. Remove the supernatant, add 300 μl PBS again, and repeat the above operation. Remove the supernatant, add 40 μl PBS to resuspend, and obtain the total protein secreted in the supernatant by the bacteria. After the cells collected by centrifugation were added to the Loading buffer, they were boiled at 100 degrees for 10 minutes to obtain the total protein of the cells. The collected total protein was detected by Western blotting (WB) method to detect the production and secretion of the target protein. Rabbit monoclonal HA-tagged antibody was used as the primary antibody, and HRP-conjugated goat anti-rabbit IgG antibody was used as the secondary antibody.

The detection results showed that Ah-JP-RBD recombinant attenuated Salmonella could effectively express and secrete RBD protein (Fig. 3A). For the four recombinant attenuated Salmonella species, Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD, and Ah-BJ-RBD, no RBD protein was found in the four bacteria when the bacteria were present in liquid LB. Ah-JJ-RBD and Ah-BJ-RBD recombinant attenuated Salmonella effectively expressed RBD protein with signal peptide when the bacteria were in macrophages and stimulated by the intracellular environment. (larger band), and after the protein is efficiently secreted into the cell interior, the signal peptide is cleaved off (smaller band). However, the expression and secretion of RBD protein could not be effectively achieved in the two engineered bacteria, Ah-NS-RBD and Ah-SS-RBD, so these two engineered bacteria were discarded in the subsequent experiments (Fig. 3B).

The expression and secretion of antigenic proteins in Ah-JJ-RBD and Ah-BJ-RBD recombinant bacteria were analyzed by immunofluorescence. After the Ah-BJ-RBD recombinant RAW264.7 (M1) cells climbed the slides, they were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 30 minutes, and washed with PBS three times. Wells were punched with 0.5% TritonX-100 in PBS for 30 minutes at room temperature. Washed 3 times with PBS, blocked with 3% BSA for 30 minutes at room temperature, washed 3 times with PBS, and incubated the slides overnight at 4 degrees using rabbit monoclonal HA-tagged antibody as the primary antibody. After washing 3 times with PBST, use monkey anti-rabbit fluorescent secondary antibody and Salmonella fluorescent antibody to incubate at room temperature for 1 hour in the dark. After washing 3 times with PBST, the nuclear dye DAPI was added and photographed using a fluorescence microscope. The results of fluorescence imaging showed that Ah-JJ-RBD or Ah-BJ-RBD recombinant attenuated Salmonella in macrophages could effectively express and secrete RBD-HA protein, but the recombinant attenuated Salmonella carrying the empty plasmid could not be detected HA-related fluorescence signal (Figure 4).

Example 4

Immunization procedures and methods

Female C57BL/6 mice aged 6-8 weeks were divided into groups of 4-5 mice per group, and each mouse was inoculated with empty bacteria or Ah-JP-RBD, Ah-JJ-RBD and Ah according to the oral dose of 1×109 CFU bacteria. -BJ-RBD 3 strains of recombinant attenuated Salmonella, once a week at an interval, and blood collection from the eyeball one week after the third time to detect the concentration of specific antibodies in the serum (Figure 5).

Example 5

RBD protein antibody detection

Preparation of immune serum: After obtaining the blood of the immunized mice according to the above method, let stand for 2-4 hours at room temperature, 3,000 rpm at 4 degrees, centrifuge for 15 minutes, collect serum and store at -80 degrees for future use.

Determination of total IgG antibody titer by ELISA: 1. Antigen coating, use 50mM, pH 9.6 carbonate buffer to dilute the recombinant S protein antigen of SARS-CoV-2 at 10μg/mL, drop 100μL per well, 4 degrees Covered with ELISA plate overnight, washed three times with PBST (every 5 minutes); 2. Blocked, added 300 μL of 3% BSA to each well of the coated plate, blocked for 3 hours at 37 degrees, and washed three times with PBST (every 5 minutes); 3. Add sample, add serum gradient dilution (50-400 times) with 100 μL PBS into the well, react at 37°C for 1-2 hours, wash three times with PBST (every 5 minutes); 4. Secondary antibody, add HRP (horseradish peroxidation) Goat anti-mouse IgG secondary antibody (1:5000 dilution in PBST) conjugated with enzyme), react at 37°C for 1 hour, wash three times with PBST (every 5 minutes); 5. Color development, add TMB substrate solution after washing the plate After 100 μL of room temperature color development for 5-10 minutes, when the color turns blue, add 100 μL of 2M concentrated sulfuric acid to each well to stop, and measure the absorption light of 450nM.

The results of antibody titer test showed that three recombinant attenuated Salmonella species, Ah-JP-RBD, Ah-JJ-RBD and Ah-BJ-RBD, could effectively induce the body to produce the corresponding RBD protein antibody, among which the Ah-BJ-RBD recombinant strain The induction effect was better (Fig. 6), indicating the stronger application value of the system.

The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and the descriptions in the above-mentioned embodiments and the description are only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have Various changes and improvements, the claimed scope of the present invention is defined by the appended claims, description and their equivalents.

Claims

A novel coronavirus vaccine antigen presentation system that attenuated Salmonella secretes and expresses RBD structural protein, characterized in that: the novel coronavirus vaccine antigen presentation system that attenuated Salmonella secretes and expresses RBD structural protein comprises type III secretion system Promoter and signal peptide sequences; can achieve the secretory expression of novel coronavirus antigens in antigen-presenting cells through attenuated Salmonella, and induce the body to produce high-titer antibodies in a mouse model.
The novel coronavirus vaccine antigen presentation system of attenuated Salmonella secreting and expressing RBD structural domain protein according to claim 1, is characterized in that: utilizes the antigen presenting cell intracellular inducible promoter to regulate the antigen secreted and expressed of bacterial secretion signal, and utilizes The Salmonella secretion expression system enables the antigen to be secreted, and the addition of a plasmid anti-loss element improves the plasmid stability of the expression vector in Salmonella, so as to obtain efficient, stable, and intracellular regulation of antigen-presenting cells. The antigen presentation of attenuated Salmonella secretion and expression system.
The novel coronavirus vaccine antigen presentation system of the attenuated Salmonella secreting and expressing RBD domain protein according to claim 1 is characterized in that: the novel coronavirus vaccine antigen is a novel coronavirus antigen table that has been screened and can induce an optimal immune response. The combination of epitopes, specifically, the epitope combination of the novel coronavirus that can induce the best immune response is the RBD structural domain of the SARS-CoV-2 spike protein, and the gene sequence of the RBD structural domain is shown in SEQ ID No.6. The nucleotide sequence shown is located at amino acids 319-541 of the overall amino acid sequence of the S protein.
The novel coronavirus vaccine antigen presentation system of attenuated Salmonella secreting and expressing RBD domain protein according to claim 1, is characterized in that: the intracellular inducible promoter of the antigen presenting cell is the Salmonella sifB promoter; the Salmonella sifB start The gene sequence of the son is the nucleotide sequence shown in SEQ ID No.4.
The novel coronavirus vaccine antigen presentation system of attenuated Salmonella secreting and expressing RBD domain protein according to claim 1, it is characterized in that: the intracellular inducible promoter of antigen presenting cell is Escherichia coli NirB promoter or Salmonella SseA start or Salmonella SseJ promoter; the gene sequence of the E. coli sifB promoter is the nucleotide sequence shown in SEQ ID No.1, and the gene sequence of the Salmonella SseA promoter is shown in SEQ ID No.2 Nucleotide sequence, the gene sequence of the Salmonella SseJ promoter is the nucleotide sequence shown in SEQ ID No.3.
The novel coronavirus vaccine antigen presentation system of attenuated Salmonella secreting and expressing RBD domain protein according to claim 1, is characterized in that: the bacterial secretion signal secretion expression system is Salmonella type III secretion expression system, and the III type secretion signal is Salmonella virulence island 2 effector protein SseJ signal peptide, and the gene sequence of the Salmonella virulence island 2 effector protein SseJ signal peptide is the nucleotide sequence shown in SEQ ID No.5.
The novel coronavirus vaccine antigen presentation system of attenuated Salmonella secreting and expressing RBD structural domain protein according to claim 1, is characterized in that: described plasmid loss prevention element is AT element sequence, and the gene sequence of described AT element sequence is The nucleotide sequence shown in SEQ ID No. 8.
The novel coronavirus vaccine antigen presentation system of the attenuated Salmonella secreting and expressing the RBD domain protein according to claim 1 is characterized in that: the expression of the vaccine antigen is realized by the attenuated Salmonella, and the attenuated Salmonella is VNP20009, htrA gene Defective attenuated Salmonella VNP20009.
The application of the novel coronavirus vaccine antigen presentation system of attenuated Salmonella secreting and expressing RBD domain protein according to any one of claims 1 to 8, it is characterized in that: the novel coronavirus vaccine antigen presentation system containing attenuated Salmonella secreting and expressing RBD domain protein by oral administration Attenuated Salmonella from a coronavirus vaccine antigen presentation system induces the production of high titer antibodies in a mouse model.
The application of the novel coronavirus vaccine antigen presentation system of the attenuated Salmonella secreted and expressed RBD domain protein according to any one of claims 1 to 8 as a medicine for preparing novel coronavirus vaccine and preventing novel coronavirus.